This invention relates to nucleic acid probes for sequence-specific recognition and cleavage of double-helical nucleic acids through the intermediacy of a triple-helix.
The sequence-specific cleavage of double-helical deoxyribonucleic acid (hereafter xe2x80x9cDNAxe2x80x9d) by naturally occurring restriction endonucleases is essential for many techniques in molecular biology including gene isolation, DNA sequence determinations chromosome analysis, gene isolation and recombinant DNA manipulations. Other applications include diagnostic reagents to detect pathogens and aberrant DNA molecules as well as chemotherapeutics.
The usefulness of DNA cleavage by these naturally recurring restriction enzymes is limited. The binding site sizes of naturally occurring restriction enzymes are typically in the range of four to eight base pairs, and hence their sequence specificities may be inadequate for mapping genomes (105-107 base pairs) over very large distances. For unique recognition of DNA in the 105-107 base pair range, sequence specificities at the 8-15 base pair level must be obtained. In addition, there are a limited number of known restriction endonucleases. Thus, they cannot be used to specifically recognize a particular piece of DNA (or RNA) unless that piece of DNA contains the specific nucleic acid sequences recognized by the endonucleases.
With the advent of pulsed field gel eloctrophoresis, separation of large (up to at least one million base pair) pieces of DNA is now possible. The design and synthesis of sequence-specific DNA recognition and cleaving molecules that go beyond the specificities of the natural restriction enzymes is obviously desirable, as they would provide valuable tools for further research, diagnostics, and chemotherapeutics.
Synthetic sequence-specific binding moieties for double-helical DNA that have been studied are typically coupled analogs of natural products (P. D. Dervan, Science 232, 464 (1986)), transition metal complexes (J. K. Barton, Science 233, 727 (1986)), and peptide fragments derived from DNA binding proteins (J. Sluka, et al., Science, in press). Additionally, methidium-propyl-EDTA (hereafter xe2x80x9cMPExe2x80x9d), which contains the metal chelator ethylenediaminetetraacetic acid (xe2x80x9cEDTAxe2x80x9d) attached to the DNA intercalator methidium, has been shown to cleave double-helical DNA efficiently in a reaction dependent on ferrous iron (Fe(II)) and dioxygen (O2). This mechanism is thought to occur by binding in the minor groove of the right-handed DNA helix. Addition of reducing agents such as dithiothreitol (hereafter xe2x80x9cDTTxe2x80x9d) increases the efficiency of DNA cleavage, as reported by Hertzberg and Dervan, J. Am. Chem. Soc. 104, 313-315 (1982); and Hertzberg and Dervan, Biochemistry. supra). MPE-Fe(II) cleaves DNA in a relatively non-sequence specific manner, and with significantly lower sequence specificity than the enzyme DNAseI, and therefore is useful in experiments to identify binding locations of small molecules such as antibiotics, other drugs, and proteins on DNA, Hertzberg and Dervan, Biochemistry, supra.
The most sequence-specific molecules characterized so far, with regard to the natural product analog approach is bis(EDTA-distamycin) fumaramide which binds in the minor grove and cleaves at sites containing nine base-pair (hereafter xe2x80x9cbpxe2x80x9d) of contiguous A,T DNA (Youngquist and Dervan, J. Am. Chem. Soc. 107, 5528 (1985)). A synthetic peptide containing 52 residues from the DNA binding domain of Hin protein with EDTA at the amino-terminus binds and cleaves at the 13 bp Hin site (Bruist, et al., Science 235, 777 (1987); Sluka, et al., supra). Another known DNA cleaving function involves the attachment of a DNA-cleaving moiety such as a ethylenediaminetetraacetic acid-iron complex (hereafter xe2x80x9cEDTA-Fe(II)xe2x80x9d), to a DNA binding molecule which cleaves the DNA backbone by oxidation of the deoxyribose with a short-lived diffusible hydroxyl radical (Hertzberg and Dervan, Biochemistry 23, 3934 (1984)). The fact that the hydroxyl radical is a relatively non-specific cleaving species is useful when studying recognition, because the cleavage specificity is due to the binding moiety alone, not some combination of cleavage specificity superimposed on binding specificity.
Despite this progress, the current understanding of molecular recognition of DNA is still sufficiently primitive that the elucidation of chemical principles involved in creating specificity in sequence recognition at the xe2x89xa715 base pair level has been slow in development in comparison to the interest in the field for mapping large genomes.
Recognition of single-stranded nucleic acids by nucleic acid-hybridization probes consisting of sequences of DNA or RNA are well known in the art. Typically, to construct a DNA hybridization probe, selected target DNA is obtained as a single-strand and copies of a portion of the strand are synthesized in the laboratory and labeled using radioactive isotopes, fluorescing molecules, photolytic dyes or enzymes that react with a substrate to produce a color change. When exposed to complementary strands of target DNA, the labeled DNA probe binds to (hybridizes) its complementary single-stranded DNA sequence. The label on the probe is then detected and the DNA of interest is thus located. Probes may similarly be used to target RNA sequences. DNA probes are currently well known in the art for locating and selecting genes of known sequence, and in the diagnosis and chemotherapy of genetic disorders and diseases.
Oligonucleotides (polynucleotides containing between 10 and 50 bases) equipped with a DNA cleaving moiety have been described which produce sequence-specific cleavage of single-stranded DNA. Examples of such moieties include oligonucleotide-EDTA-Fe hybridization probes (xe2x80x9cDNA-EDTAxe2x80x9d) which cleaves the complementary single strand sequence (Dreyer and Dervan, Proc. Natl. Acad. Sci. USA. 82, 968 (1985); Chu and Orgel, Proc. Natl. Acad. Sci. USA. 82, 963 (1985)). Such probes are disclosed in the co-pending U.S. Patent Application of Dervan et al., xe2x80x9cNucleic Acid Probes And Method For Using Same,xe2x80x9d Ser. No. 695,082, filed Jan. 25, 1985, and assigned to the same assignee as the present application.
In addition to double and single-stranded configurations, it is also well known in the art that triplexes of nucleic acids naturally exist (Howard, et al., Biochem. BioPhys. Res. Commun. 17, 93 (1964)). Poly(U) and poly(A) were found to form a stable 2:1 complex in the presence of MgCl2. After this, several triple-stranded structures were discovered (Michelson, et al., Proc. Nucl. Acid Res. Mol. Biol. 6, 83 (1967); Felsenfeld and Miles, Annu. Rev. Biochem. 36, 407 (1967)). Poly(C) forms a triple-stranded complex at pH 6.2 with guanineoligoribonucleotides. One of the pyrimidine strands is apparently in the protonated form (Howard, et al., supra). In principle, isomorphous base triplets (T-A-T and C-G-C+) can be formed between any homopyrimidine-homopurine duplex and a corresponding homopyrimidine strand (Miller and Sobell, Proc. Natl. Acad. Sci. U.S.A. 55, 1201 (1966); Morgan and Wells, J. Mol. Biol. 37, 63 (1968); Lee et al., Nucleic Acids Res. 6, 3073 (1979)). The DNA-duplex poly(dT-dC)-poly(dG-dA) associates with poly(U-C) or poly(dT-dC) below pH 6 in the presence of MgCl2 to afford a triple-stranded complex. Several investigators have proposed an anti-parallel orientation of the two polypyrimidine strands based on an anti conformation of the bases, ibid. X-ray detraction patterns of triple-stranded fibers (poly(A)-2poly(U) and poly(dA)-2poly(dT)) supports this hypothesis (Arnott and Bond, Nature New Biology 244, 99 1973); Arnott and Selsing, J. Mol. Biol. 85, 509 (1974); and Arnott et al., Nucleic Acids Res. 3, 2459 (1976)), and suggested an Axe2x80x2-RNA-like conformation of the two Watson-Crick base paired strands with the third strand in the same conformation, bound parallel to the homopurine strand of the duplex by Hoogsteen-hydrogen bonds. (Hoogsteen, Acta Cryst. 12, 822 (1959)). The twelve-fold helix with dislocation of the axis by almost three angstroms, the C3xe2x80x2-endo sugar puckering and small base-tilts result in a large and deep major groove that is capable of accommodating the third strand (Saenger, Principles Of Nucleic Acid Structure, edited by C. R. Cantor, Springer-Verlag, New York, Inc. (1984). A high resolution X-ray structure of a triple-helical DNA or RNA is not known in the art. Importantly, there are no techniques described in the literature for determining whether a specific homopyrimidine-homopurine tract (e.g. 15 bp) within a large duplex DNA (e.g.  greater than 103 bp can form a triple helix as a method of recognition at that site.
No analytical or recombinant DNA applications of triple-helical DNA or RNA have been reported. Although triple-stranded structures of polynucleotides were discovered decades ago, the biological significance has remained obscure. Such triplexes were proposed to be involved in processes such as regulation of gene expression, maintenance of folded chromosome conformations, chromosome condensation during mitosis, and induction of local conformational changes in B-DNA (Morgan, Trends Biochem. Sci. 4, N244 (1979); Hopkins, Comments Mol. Cell Biophys. 2, 153 (1984); Minton, J. Exp. Path. 2, 135 (1985)).
The above-described methods for sequence-specific DNA recognition and cleavage have been limited to single-stranded DNA hybridization probes, to natural or synthetic restriction endonucleases, and to those molecules which recognize sequences of DNA directly such as antibiotics, and DNA intercalators such as methidium.
Surprisingly, the present inventors have discovered compositions and methods of specifically tailored recognition of a significantly larger number of double-stranded DNA and RNA sites than was previously possible, utilizing triple helix formation (xe2x89xa715 bp recognition) at discrete highly specific sites within large DNA. The compositions of the present invention, utilized by the methods set forth herein, will provide useful tools for chromosome analysis, gene mapping and isolation. Moreover, as molecular biology defines specific disease states at the DNA level, the present invention finds usefulness in diagnostic strategy, as well as chemotherapeutics.
The present invention relates to recognition of homopyrimidine-homopurine double-helical tracts within large DNA, RNA, and in DNA-RNA double-helical hybrid duplexes, by triple-helix formation under physiologic conditions. The present invention also relates to cleavage of said discrete, double-helical tracts.
One object of the present invention is to provide homopyrimidine oligonucleotides and their neutral or cationic analogs equipped with markers, lables, chemotherapeutic agents and/or efficient DNA cleaving moieties at the 5xe2x80x2 end, which are capable of forming triple helices, which can be produced in sufficient quantities to provide pharmaceutical, laboratory, or industrial compositions useful for chromosome analysis, gene mapping and isolation, diagnostics and chemotherapeutics. An additional object of the present invention is to utilize precisely tailored polynucleotide hybridization probes adapted for automated synthesis and which afford control over the precise location in a large double-helical nucleic acid of a label or DNA cleaving moiety at any base position in the polynucleotide probe strand.
Another object of the present invention is to provide polynucleotide hybridization probes and methods for their use in the recognition of any specific sequence within a large double-helical nucleic acid. Such probes are designed and adapted as described above, with the substitution of a radioactive label, photolytic dye, enzyme, or a fluorescing, or otherwise detectable molecule for the DNA cleaving moiety.
One object of this invention is to provide a method for delivering chemotherapeutic agents in vivo that eliminates the need to denature the DNA before the agent can act. Yet another object of this invention is to provide a method for precisely locating a chemotherapeutic agent or replacement gene sequence at a specific homopyrimidine-homopurine tract anywhere in a large double-stranded nucleic acid. This invention also finds application in diagnostics for gene-based diseases, and eliminates the need for many steps in the commonly used diagnostic processes.
It is also an object of the present invention to provide a new assay for triple helices.
Additional objects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description or may be learned from practice of the invention. The objects and advantages may be realized and obtained by means of the instrumentalities and combinations particularly pointed out in the appended claims.
To achieve the objects and in accordance with the purposes of the present invention, homopyrimidinepolydeoxyribonucleotide probes with at least one detectable marker, chemotherapeutic agent or a DNA-cleaving moiety attached to at least one predetermined position are set forth which are capable of binding the corresponding homopyrimidine-homopurine tracts within large double-stranded nucleic acids by triple-helix formation at a predetermined site.
The compositions and the methods of the present invention allow for cleavage of one or both strands of the Watson-Crick DNA. It is this cleavage event by bifunctional DNA-EDTA probes (i.e., recognition and cleavage) that allowed the triple helix formation at discrete locations to be mapped on large DNA using gel electrophoresis.
The polynucleotide sequences of the invention may be either synthetic sequences or restriction fragments (xe2x80x9cnaturalxe2x80x9d) DNA sequences. The compositions and methods of this invention are understood to apply equally for double-helical RNA, as well as to hybrid duplexes with one strand of DNA and one strand of RNA.
Also to achieve the objects of this invention, an assay for triple helices of up to at least 15 bp is disclosed.
Additionally, to achieve the objects and in accordance with the purposes of the present invention, a method is disclosed which results in recognition, chemotherapeutic alteration, and if desired, cleavage of homopyrimidine-homopurine tracts within a large double-stranded nucleic acid by triple helix formation at a particular, predetermined site. This method comprises:
(a) hybridizing a specific homopyrimidine-homopurine tract within a large double-stranded nucleic acid with a corresponding polynucleotide hybridization probe, said nucleotide containing at least one nucleoside to which is attached at least one of the following:
(i) at least one detectable label molecule capable of being detected upon the binding of said nucleotide to said tract,
(ii) at least one molecule adapted to cleave at least one strand in said homopyrimidine-homopurine tract,
(iii) at least one molecule of a chemotherapeutic agent;
(b) permitting said hybridization to proceed to formation of a triple-helix; and
(c) at least one of the following:
(i) detecting said label;
(ii) cleaving one or both strands of the nucleic acid; or
(iii) permitting said chemotherapeutic agent to act.
It is understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.